Metastatic tumor cells are distinguished by their ability to invade the basement membrane of epithelial barriers and migrate to distant sites. Recent studies from this program demonstrate that host macrophages are critical for the motility and invasion of tumor cells, due to a paracrine loop involving the mutual signaling and chemotaxis between macrophages and tumor cells. PI3K is a critical regulator of cell motility, and distinct PI3K isoforms are required for regulation of actin-based motility in tumor cells versus macrophages. We propose to use isoform-specific inhibitors of Class IA PISKs as well as genetic approaches to examine the requirement for PI3K-mediated motility of both tumor cells and macrophages during invasion and metastasis. By selectively inhibiting the motility of tumor cells versus macrophages, we will test whether the enhanced tumor cell chemotaxis observed in the presence of macrophages requires pre-exposure to macrophage-derived cytokines, versus the presence of continuous macrophage signaling during coordinated migration of the two cell types. We will also examine the metastatic behavior of tumor cells expressing activating mutations of PI3K that are commonly found in human breast cancer. The unique assays developed by this program will allow a detailed analysis of how oncogenic p110ct mutants affect the tumor cell-macrophage paracrine loop. Finally, studies from this program have shown that genes coding for proteins that modulate the myosin-ll regulatory pathway are up-regulated in invasive tumor cells and that the amoeboid motility of tumor cells in a 3D matrix is mediated by the myosin-ll regulatory pathway. Given these findings, and our observations that PI3K regulates myosin-ll-based contractility in tumor cells, we will examine the PI3K isoform-dependence of myosin-ll phosphorylation, the identification of intermediary signaling pathways in tumor cells and macrophages, and the subsequent effects on motility and invasion. These studies will complete our analysis of the motility cycle as it relates to the invasion signature, and lead to new insights into the role of macrophage-tumor cell paracrine signaling during metastasis.